Although much progress has been made in detecting and treating cancer, disseminated metastatic disease remains the leading cause of death in cancer patients. Frustratingly little is known regarding how these deadly lesions respond to and eventually resist treatment in vivo due to their widespread nature, often sub-clinical sizes, and highly heterogeneous microenvironments. The lack of appropriate imaging tools to understand and overcome treatment resistance on the microscale in metastatic cancer represents a major unmet need in both cancer research and therapeutics. To address this critical challenge, this application introduces an innovative microscopic technological paradigm for visualizing and understanding the microscale treatment response and resistance factors found in disseminated metastatic cancer. The goal of this work is to develop a high- throughput, live-cell, molecular and structural optical microscopy platform capable of visualizing and monitoring therapeutic response in micrometastatic lesions in vitro at both the cellular and nodular levels. Such an imaging platform will enable a bottom-up approach for basic cancer and cancer therapeutics research by building a detailed spatiotemporal picture of cancer therapy from single cells all the way to the whole tumor. Based upon the complementary molecular and structural imaging technologies of hyperspectral microscopy and optical coherence tomography (OCT), the automated multiplexed therapy-imaging platform will enable long-term cellular and nodular-level studies that map the 3D distribution of molecular treatment factors within a structural context. The new real-time, tunable, near-IR optimized hyperspectral microscopy system will allow for 3D, multi-fluorophore, live-cell imaging of treatment resistance factors deep in tumor nodules. For longitudinal microscale visualization of the complex structural changes caused by treatment, the non- perturbative and label-free time-lapse OCT (TL-OCT) modality will be integrated along with the hyperspectral microscope. Applicable to many metastatic cancers, this new imaging platform will be validated using a physiologically relevant, repeatable, and multiplexed in vitro 3D model of micrometastatic ovarian cancer for imaging-based screens. In the first application of this approach, the multimodal technological platform for cancer biology research will focus on visualizing and correlating several crucial treatment resistance factors, such as drug uptake and diffusion, hypoxia, and pH, with treatment response. This imaging-based approach to systematically and quantitatively characterize cancer therapy response and resistance represents a departure from traditional methods that miss crucial molecular and structural information. These technologies can be readily translated for imaging patient tissue samples ex vivo, or in situ microendoscopically, for image-guided therapeutic planning. By building our knowledge of treatment response and resistance at the microscale, this innovative imaging platform forms the foundation of a transformative, new investigator-led research program aiming to improve current cancer therapeutics and defeat treatment-resistant metastatic disease. PUBLIC HEALTH RELEVANCE: Cancer that becomes metastatic is too often fatal due to the spread of numerous microscopic lesions that grow to be treatment resistant. The goal of this application is to address the root causes of treatment resistance in cancer through an innovative microscale imaging approach capable of visualizing the crucial molecular and structural factors involved in therapeutic response. By building an understanding of how deadly lesions escape therapy from the cellular level to the whole tumor, and using this knowledge to defeat treatment-resistant disease, the technology developed in this application aims to significantly improve the lives of patients suffering from advanced metastatic cancer.